Abuse of cocaine is an intractable social and medical problem that is resistant to remediation through pharmacotherapy. Cocaine acts to block the reuptake of monoamines, dopamine, norepinephrine, and serotonin thus prolonging and magnifying the effects of these neurotransmitters in the central nervous system (Benowitz, 1993). Cocaine toxicity is marked by both convulsions and cardiac dysfunction (e.g., myocardial infarction, cardiac arrhythmias, increased blood pressure, stroke, or dissecting aneurysm, and increased myocardial oxygen demand), due to effects on neurotransmitter systems and myocardial sodium channel blockade (Bauman and DiDomenico, 2002; Wilson and Shelat, 2003; Knuepfer, 2003). Because of cocaine's ability to readily cross the blood brain barrier and its widespread effects on the central and peripheral nervous systems, overdose can result in sudden death (see Bauman and DiDomenico, 2002 for review).
Although the mechanism of cocaine's action is well understood, this information has not yet resulted in the development of an effective antagonist of cocaine that could be used in abuse and overdose situations. The rapid and pleiotropic effects of cocaine present a complex problem for the treatment of acute cocaine toxicity (Carroll and Kuhar, 1999). The two types of therapies that are available for the treatment of opioid abuse, antagonism (e.g., naltrexone) and replacement (e.g., methadone), do not have parallels in the case of cocaine, although attempts at the latter are being considered (e.g., Grabowski et al., 2004). One approach is to prevent or reduce the cocaine from reaching sites of action by administering either endogenous esterases, cocaine specific antibodies, or a catalytic antibody.
Naturally occurring cocaine is hydrolyzed at the benzoyl ester by serum butyrylcholinesterase (BChE) to nontoxic ecgonine methyl ester and benzoic acid. In the liver, carboxylesterase hCE-2 hydrolyzes the methyl ester to yield benzoylecgonine and methanol. The elimination half-life of cocaine in the blood ranges from 0.5 to 1.5 hr (Inaba, 1989). There have been a few attempts to use naturally occurring BChE or genetically engineered BChE to increase cocaine breakdown (see, e.g., Carmona et al., 2000; Xie et al., 1999; Sun et al., 2002a; Sun et al., 2002b; Duysen et al., 2002; Gao and Brimijoin S, 2004; Gao et al., 2005). Other researchers have utilized a monoclonal antibody, Mab 15A10, as a catalytic antibody to cocaine (see e.g., Landry et al, 1993; Mets et al., 1998), while others are exploring the use of cocaine vaccines (see e.g., Kosten et al., 2002).
A bacterium, Rhodococcus sp. MB 1, indigenous to the soil surrounding the coca plant, has evolved the capacity to utilize cocaine as its sole carbon and nitrogen source. The bacterium expresses a cocaine esterase (CocE) that acts similarly to BChE to hydrolyze the benzoyl ester of cocaine, yielding ecgonine methyl ester and benzoic acid (FIG. 1) (Bresler et al., 2000; Turner et al., 2002; Larsen et al., 2002). The gene for CocE has been isolated and cloned (Bresler et al., 2000), and the crystal structure of CocE has been determined (Turner et al., 2002; Larsen et al., 2002).
The purified enzyme (MW ˜65 kDa) catalyzes cocaine very efficiently with Michaelis-Menten kinetics kcal=7.2 s−1 and Km=640 nM (Turner et al., 2002; Larsen et al., 2002), nearly three orders of magnitude greater than endogenous esterases and, most likely, would act quickly enough to detoxify humans who have overdosed on cocaine (Landry et al., 1993; Mets et al., 1998). Additionally, the esterase also metabolizes cocaethylene, a potent metabolite of cocaine and alcohol, almost as efficiently as it metabolizes cocaine (kcat=9.4 s−1 and Km=1600 nM) (Turner et al., 2002; Larsen et al., 2002).
One aspect of the Rhodococcus CocE that limits its usefulness is its low thermostability—its t1/2 at 37° C. is about 15 minutes, whereas its t1/2 at 4° C. is >6 mo (PCT Patent Application PCT/US2007/015762, incorporated by reference herein). Thermostability was genetically engineered into CocE, with several mutant proteins having an increased t1/2 at 37° C. up to ˜326 min (Id.).
There is a need for additional methods and compositions for thermostabilization of CocE. The present invention addresses that need.